QUANTITATIVE ELECTRON PROBE ANALYSIS OF HAEMOLYMPH AND URINE COMPOSITION FROM INSECT CALLIPHORA ERYTHROCEPHALA DURING METAMORPHOSIS

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QUANTITATIVE ELECTRON PROBE ANALYSIS OF HAEMOLYMPH AND URINE COMPOSITION FROM INSECT CALLIPHORA ERYTHROCEPHALA

DURING METAMORPHOSIS

W. Alkassis, N. Roinel, N. Koechlin

To cite this version:

W. Alkassis, N. Roinel, N. Koechlin. QUANTITATIVE ELECTRON PROBE ANALYSIS OF

HAEMOLYMPH AND URINE COMPOSITION FROM INSECT CALLIPHORA ERYTHRO-

CEPHALA DURING METAMORPHOSIS. Journal de Physique Colloques, 1984, 45 (C2), pp.C2-

481-C2-484. �10.1051/jphyscol:19842109�. �jpa-00223776�

QUANTITATIVE ELECTRON PROBE ANALYSIS OF HAEMOLYMPH AND URINE

COMPOSITION FROM INSECT CALLIPHORA ERYTHROCEPHALA DURING METAMORPHOSIS

W. A l k a s s i s , N. Roinel* and N. Koechlin**

Laboratoire de Physiologie des Insectes, Universite P. et M. Curie, 75005 Paris, France

Departement de Biologie, Labovatoire de Physiologie Physioo-Chimique, C.E.N.

Saolay, 91191 Gif-sur-Yvette, France

**Laboratoire d'Eistologie et Cytologie des Invertebres Marins, Universite P.

et M. Curie, 75005 Paris, France

Résumé - Les teneurs en Na, K, Mg, Ca et P de l'urine distale et de l'hémolymphe de la mouche Calliphora e. ont été mesurées au cours de la métamorphose à l'aide d'un analyseur à sonde électronique.

Abstract - Na, K, Mg, Ca and P concentrations in distal urine and haemolymph of the blowfly Calliphora e. have been measured during metamorphosis. Quantitative analysis of liquid microsamples has been performed with an electron probe analyser.

Microtechniques of analysis were initiated by RAMSAY (1) who applied them to the understanding of the physiology of excretion and osmoregulation of small invertebrates like insects. Nowadays the electron probe analysis allows accurate elemental concentration measurements of insect internal fluids (2) or of insect transporting epithelia (3). Data have been obtained from fluids pooled from several Drosophila hyder (2) and from samples from individual Heteroptera (4). Results always concerned one animal stage, either the adult (=

imago) or the larvae stage. However very l i t t l e is known about the changes in ion composition and excretion of the insect during metamorphosis. The Malpighian tubules of Diptera are the only organs to keep a permanent structure during metamorphosis, although their intracellular organization and their size are modified (5). In the present work, samples of distal urine and haemolymph from individual Calliphora erythrocephala were analysed at several stages of metamorphosis. The concentrations of the elements contained in both fluids were determined with an electron probe analyser (6).

Material and Methods

The different stages examined were : three day old larvae (L3), 2, 5 and 7 day old pupae (P2, P5, P7) and imago (I). In each stage, eight laboratory-bred animals were used.

Haemolymph micropuncture samples were obtained and kept under water-saturated paraffin oil (on account of their very high protein content i t was not possible to prepare samples of haemolymph from P5 for probe analysis). Distal urine contains a great number of spherical concrements. The analyses were performed upon both liquid and concrements, the latter being dissolved prior to analysis in 4% TCA and the results corrected for this dilution. The volumes analysed were 0.3 nl. The electron probe analyser was a Cameca MBX adjusted as follows: 15 kV, 300 nA, beam diameter 80 )im,

Results

Haemolymph and distal urine concentrations values (in mmoles/1) are given for each stage studied in table I and Figs. 1, 2 and 3.

In haemolymph, Na, Mg and Ca decreased significantly from 116, 14 and 6 mmol/1 respectively (in 1_3) to 58, 7 and 1 (in P2), then increased again to 140, 4 and 4 (in I). K and P increased significantly from 11 and 18 (in L3) to 39 for both (in P7) and decreased to 23 and 32 (in I).

In distal urine, a large increase in the K, Mg, Ca and P concentrations was observed

during the three first days of metamorphosis (from L3 to P2). Mg, Ca, P and K concentrations

increased significantly from 560, 210, 370 and 140 mmoles/1 (in L3) to 3000, 1500, 4400 and

430 (in P2). Between P2 and P5 no significant change occurred. The values for Ca and P for the

final stage (I) were similar to those found in L3, while the values for K and Mg were two to

Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:19842109

C2-482 JOURNAL DE PHYSIQUE

Stages L3

TabIe I : Na, K, Ca, Mg and P distal urine (U) and haemolymph (HI concentrations during metamorphosis. Mean value + ^SEM.

Ca 7 Haemolymph

M . -- 1

E

-

I00

50

-

L3 P2 p7 l M AGO

Fig. 1 : Na, K, Mg, Ca and P haemolymph concentrations during metamorphosis.

t h r e e t i m e s lower in t h e s t a a e I than in s t a a e L3. Na d e c r e a s e d sianificantlv f r o m 2800 (in L3) - -

t o 500 (in I).

Thus t h e d i s t a l urine c o n c e n t r a t i o n of everv analvsed e l e m e n t w a s invariablv hiaher

t h a n t h e corresponding haernolymph concentration, i r r e s p e c t i v e of t h e s t a g e involved.

Discussion

T h e g e n e r a l tissue histolysis taking p l a c e during t h e f i r s t t h r e e days ( f r o m L 3 t o P2) induces t h e r e l e a s e of t h e ionic c e l l components. Among t h e m , Ca, Mg and P a r e strongly drawn t o w a r d s Malpighian tubules and a c c u m u l a t e d in distal urine. Such a n a c c u m u l a t i o n might explain t h e i n c r e a s e in s i z e and number of t h e d i s t a l urine c o n c r e m e n t s in which Ca, Mg and P c o n t e n t is very high (5). Alkaline e a r t h precipitation in c o n c r e m e n t s could b e induced by a n acid-phosphatase rich organic phase (2). During t h e s a m e period (from L 3 t o P2) haemolymph is d e p l e t e d in C a and Mg. The c o n c e n t r a t i o n s of t h e s e ions, which a r e low c o m p a r e d t o t h o s e in t h e l a r v a e and imago, a r e probably conducive t o t h e f o r m a t i o n s of new t i s s u e s - o n t h e c o n t r a r y , i t s e e m s t h a t high P and K c o n c e n t r a t i o n s in haemolymph during t h e s a m e period a r e n o t prejudicial f o r organogenesis.

Between s t a g e s P2 and P5, t h e stability of t h e c o n c e n t r a t i o n v a l u e s observed for a l l measured e l e m e n t s ( e x c e p t Na) in d i s t a l u r i n e is t h e consequence of t w o conflicting processes:

t h e end of histolysis which s t i l l yields f r e e ions and t h e s t a r t of organogenesis which r e u s e s them.

F r o m P 5 t o I, a seven o r e i g h t day period during which t h e a n i m a l s s t i l l do n o t t a k e food, e l e m e n t s supplied t o newly organized tissues c o m e f r o m haernolymph and possibly f r o m c o n c r e t i o n s in t h e d i s t a l urine. However t h e d e c r e a s e in n u m b e r a n d s i z e of t h e c o n c r e t i o n s which is observed during t h i s period may also b e d u e t o t h e i r p a r t i a l elimination in t h e urinary flow which s t a r t s a t t h i s period.

Sodium s t o r a g e during metamorphosis is n o t e f f e c t e d by t h e d i s t a l urine and f u r t h e r investigations a r e necessary t o a n s w e r t o t h i s question. As t h e Na c o n t e n t s of t h e haemolymph a r e similar f o r larvae and imago, while t h e Na c o n t e n t s of t h e i r urine d i f f e r ( t h a t of imago being lower) one concludes t h a t sodium t r a n s p o r t by t h e Malpighian e p i t h e l i a is d i f f e r e n t in t h e t w o stages. The large Mg and C a c o n t e n t d i f f e r e n c e s observed b e t w e e n l a r v a e and imago haemolymphs might b e r e l a t e d t o d i f f e r e n c e s in d i e t o r in m e t a b o l i c processes.

R e f e r e n c e s

1. RAMSAY J.A., J. exp. Biol. 2 (1953) 358.

2. HEVERT F., WOLBURG H. and WESSING A., Cytobiol. 33 (1974) 312.

C2-484 JOURNAL DE PHYSIQUE

3. GUPTA B.L., H A L L T.A. and MORETON R.B. (1977) in "Transport of Ions and Water in Animal", GUPTA B.L., MORETON R.B., OSCHMAN J.A. and WALL B.J. eds, Academic Press, London, 83.

4. STADDON B.W. and EVERTON I.J., Comp. Biochem. Physiol., 65A (1980) 371.

5. ALKASSIS W., These dlEtat, Univ. P. e t M. Curie, Paris, ~ r a n c n u i n 1983).

6. MOREL F. and ROINEL N., J. Chim. Phys. Phys. Chim., g, 6 (1969) 1084.